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Total RNA (1 μg) from wild-type and dHAND-null E9.5 hearts was treated with deoxyribonuclease and used to generate cDNA by the SMART cDNA synthesis kit (Clontech). PCR-Select (Clontech) was used for subtractive hybridization. Briefly, two pools of Rsa I-digested wild-type cDNA were used as tester and ligated to unique adapters. Rsa I-digested dHAND-null cDNA was used as driver without adapters. Hybridization of the tester population with excess driver and amplification of subtracted species with the two unique adapters in the tester cDNA produced a pool of PCR-amplified fragments theoretically present in wild-type, but not dHAND mutant, RNA. The PCR products were cloned and sequenced.
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Total RNA was purified from hearts of wild-type and dHAND-null embryos at E9.5 and used for cDNA synthesis: PCR analysis was performed with oligonucleotides specific for mouse Ufd1 (upper, 5′-GGAGAAAGGAGGGAAGATAA-3′; lower, 5′-CGAAGTAGGAGAAGTTGGAA-3′) or mouse Cdc45 (upper, 5′-ACCACTTCATCCAGGCTCTC-3′; lower, 5′-GGT- GCTTTCTGCTGCCTTCT-3′) under the following conditions: 94°C for 5 min; 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min; and 72°C for 7 min. PCR products were analyzed by agarose gel electrophoresis after serial cycles in the linear range of amplification. RNA loading was controlled for by amplification of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Negative controls were performed for each sample using non-reverse-transcribed RNA. A Southern blot of the samples was hybridized for UFD1L or G3PDH.
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Whole-mount in situ hybridizations were performed with digoxygenin-labeled antisense riboprobes synthesized from the 700-bp open reading frame and the 250-bp 3′ untranslated region of mouse UFD1L cDNA (9). Embryos were photographed with and without clearing in benzyl benzoate:methyl salicylate (1:1). For histologic analysis, stained embryos were embedded in paraffin after fixation. Transverse and sagittal sections were made at 8-μm intervals throughout the embryos.
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32P-labeled UFD1L or CDC45 cDNA.
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Thymic tissue was collected from patients who were undergoing surgical repair of CHD, and total RNA was extracted and used for cDNA synthesis. Northern analysis was performed on 20 μg of total thymic RNA and hybridized to a UFD1L cDNA probe. HIRA-and CDC45-specific amplimers were used to amplify the respective cDNAs. HIRA amplimers were: upper, 5′-GACGGCTCTGTGGCATTCCT-3′; lower, 5′-GCCATCTGCTGTCCGAGTCT-3′. CDC45 amplimers were: upper, 5′-GCCTTGTTCCAGTGTGACCA-3′; lower, 5′-GTTCTCCTCATCCTCGTTCC-3′.
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We thank R. Schultz and A. Bowcock for helpful discussions and assistance with genetic analyses, members of the pediatric cardiology and cardiothoracic surgery divisions for assistance with tissue collection, other members of the Srivastava laboratory for critical discussion, J. L. Goldstein, M. S. Brown, E. N. Olson, and H. H. Hobbs for critical review of this manuscript, and J. Page for manuscript preparation. Supported by grants to D.S. from NIH (R01HL57181-01) and March of Dimes.
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